Visualisation of in vivo protein synthesis during mycobacterial infection through [68Ga]Ga-DOTA-puromycin µPET/MRI

Radiolabelled puromycin analogues will allow the quantification of protein synthesis through nuclear medicine-based imaging. A particularly useful application could be the non-invasive longitudinal visualisation of mycobacterial activity through direct quantification of puromycin binding. This study assesses the value of [68Ga]Ga-DOTA-puromycin in the visualisation of mycobacteria through positron emission tomography combined with magnetic resonance imaging (µPET/MRI). The radiopharmaceutical was produced by previously published and validated methods. [68Ga]Ga-DOTA-Puromycin imaging was performed on severe immunodeficient mice infected with Bacille Calmette-Guérin-derived M. Bovis (BCG). Acute and chronic infection stages were examined by µPET/MRI. A follow-up group of animals acted as controls (animals bearing S. aureus-derived infection and sterile inflammation) to assess tracer selectivity. [68Ga]Ga-DOTA-puromycin-µPET/MRI images revealed the acute, widespread infection within the right upper shoulder and armpit. Also, [68Ga]Ga-DOTA-puromycin signal sensitivity measured after a 12-week period was lower than that of [18F]FDG-PET in the same animals. A suitable correlation between normalised uptake values (NUV) and gold standard histopathological analysis confirms accurate tracer accumulation in viable bacteria. The radiopharmaceutical showed infection selectivity over inflammation but accumulated in both M. Bovis and S. Aureus, lacking pathogen specificity. Overall, [68Ga]Ga-DOTA-puromycin exhibits potential as a tool for non-invasive protein synthesis visualization, albeit without pathogen selectivity.


Animal response to infection
The study effectively achieved its goals by investigating two distinct objectives.The first objective centred around tracer imaging accuracy and specificity, while the second objective focused on tracer selectivity.These investigations were conducted using two separate animal groups.Prior pilot tests were performed during the development of the animal model to determine the inoculated BCG dose needed to result in pathology of infection and successful infection was evaluated through the presence of sufficient levels of acid-fast-bacilli during staining of infected tissue ex vivo.For group 1 (N = 20) in this study, 6 mice were used after 12 weeks to confirm sufficient bacterial load in the infected lungs and percutaneous tissues.Humane endpoint euthanasia was performed in 4/20 (20%) animals due to inacceptable severity of symptoms.The remaining set of animals (n = 10) showed acceptable symptoms and fitness to be used for the acute and chronic disease state groups.Those animals tolerated the acute interventions well.Subsequently the state of disease progression was acceptable to study longterm chronic effects of BCG.All 5 animals of group 2 showed acceptable symptoms and fitness to perform the required imaging procedures.

Ga]Ga-DOTA-puromycin-µPET/MRI imaging of acute BCG infection
Animals from group 1 received a percutaneous (right front leg) BCG dose and [ 68 Ga]Ga-DOTA-puromycin-µPET/MRI imaging was performed at 24 h after inoculation.All µPET/MRI images (n = 5) clearly indicated elevated, dispersed radioactivity as displayed at the injection site of the right front leg (Fig. 1a, #1).The presence of activity in the lower bronchial tract (Fig. 1a, #3) may be attributed to the acute state after inhalation of BCG.
Results from the image-guided tissue quantification are summarised in Table 1 and correlated with the microanatomical changes as indicators of inflammation and bacilli-specific Ziehl-Neelsen staining.A normalised uptake value (NUV) of 0.69 ± 0.16 was observed for [ 68 Ga]Ga-DOTA-puromycin in the percutaneous infection site.No tracer uptake was seen in the sham inoculation area (saline) which allowed for a calculated target to non-target ratio of 2.79 ± 0.45 representing the BCG inoculation site.The BCG-derived pulmonary infection site (low density) had much lower NUV and T/NT ratios.Both NUV and T/NT ratios correlated well with the histology gradings presented as these also indicated a lower rate of infection in the pulmonary tissue compared to the inoculated area.
A parallel investigation (Group 2) was performed as a control to determine tracer selectivity for mycobacteria species above sterile inflammation or gram-positive infections (i.e., S. aureus-derived subcutaneous infection).Animals from group 2, showed clear uptake in the S. Aureus inoculation site (Fig. 1b, #4), but negligible signal for the contralateral sterile inflammation site (Fig. 1b, #6).
Results from the image-guided tissue quantification are summarised in Table 2 and correlated with the microanatomical changes as indicators of inflammation and Ziehl-Neelsen staining.At the inflammation site no noteworthy accumulation of [ 68 Ga]Ga-DOTA-puromycin was present.The NUV of the S. Aureus inoculation area was 0.78 ± 0.15 with a target to non-target ratio of 3.57 ± 0.29.This is not significantly different when compared to the BCG inoculation site indicating that the tracer is non-selective towards certain types of infections (i.e., a TB specific radiopharmaceutical).Additional ex-vivo biodistribution data and images are also included in the Supplementary Figure S3 and S4.www.nature.com/scientificreports/

Non-invasive monitoring of chronic BCG infection
The µPET/MRI images acquired 12 weeks after BCG administration (Fig. 2) clearly visualised widespread infection (in both modalities) on the right upper torso originating from the infection induced in the armpit aerial.Low density BCG foci in the lung (2nd infection site) could not be clearly identified, instead a diffuse, slightly elevated tissue signal is evident.High radioactivity in kidneys and bladder confirmed prior findings on the main excretion pathway via a renal clearance.The µPET/MRI image guided analysis of relevant tissues (Table 3) revealed high variabilities for both [ 18 F] FDG and [ 68 Ga]Ga-DOTA-Puromycin NUVs.[ 18 F]FDG NUVs were much higher for infected tissues (sevenfold for percutaneous tissue and a 13-fold for pulmonary tissue) over the healthy tissue references and twofold higher than the hepatic tracer concentration.In contrast, [ 68 Ga]Ga-DOTA-puromycin NUVs were moderate to  www.nature.com/scientificreports/low, however a fourfold higher percutaneous tissue concentration and a twofold for pulmonary tracer concentration was calculated over the healthy tissue references.The liver NUV range for [ 68 Ga]Ga-DOTA-puromycin (0.12 -0.21) was favourably lower compared to [ 18 F]FDG (0.33-0.42).The total hepatic tracer uptake was 3.51 ± 0.39%ID/g.Evidence of mycobacterial manifestation was confirmed by tissue staining with grade 4 in the percutaneous BCG infection site and grade 5 for the infected lungs (not whole lungs).Evidence of systemic mycobacterial disease was suggested by change in animal behaviour but was not confirmed by the data-no inflammation in spleen (not shown) and liver.
For signal accuracy, Spearman's rank correlation test resulted in an adequate correlation of the [ 68 Ga] Ga-DOTA-puromycin NUV to the level of mycobacterial presence ZN stain grading (Fig. 3b); but [ 18 F]FDG NUV was non-correlative.In addition, a miscorrelation between [ 18 F]FDG-and [ 68 Ga]Ga-DOTA-puromycin values was observed (Supplementary Figure S1).However, [ 18 F]FDG-PET provided approximately tenfold higher NUV values than the [ 68 Ga]Ga-DOTA-puromycin (Supplementary Figure S1).This indicates that the gallium-68 based tracer maybe more accurate but its imaging performance cannot outperform [ 18 F]FDG-PET for the quantification of pulmonary BCG-derived infection in vivo.

Discussion
PET imaging has gained global prominence as an increasingly employed technique, recognized for its robust utility in non-invasive clinical disease diagnosis, as well as in monitoring responses and assessing outcomes of drug treatments.PET is advantageous in the fact that it offers quantitative imaging of physiological function.This is of particular importance for smaller regions of interest such as infective foci.Eigner et al. have originated the radiolabelling of puromycin with the PET radionuclides scandium-44 and gallium-68 and over the past 10 years both carbon-11-and flourine-18-labeled puromycin derivatives were developed accordingly [13][14][15] .Such a development may help to provide a platform for clinical trial investigations soon, provided that the role and clinical values of radiolabelled puromycin derivatives are clearly defined.
Some factors need to be considered when using Puromycin as an infection imaging agent namely the presence of puromycin resistant organisms and secondly the non-selectivity of Puromycin for prokaryotic cells.Resistance towards puromycin takes place through enzymatic inhibition of puromycin or through alterations in cell wall permeability.However, resistance towards puromycin is highly unlikely as this is not an antibiotic that is used to treat human infections 19 .More often, it is used as a selection antibiotic for genetically engineered cell-lines or as a probe to evaluate protein synthesis 20 .
Puromycin binds non-selectively binds to ribosomes, mimicking the structure of aminoacyl-tRNA.This allows puromycin to enter the ribosome's A-site, where it is incorporated into the nascent peptide chain.Due to its structural mimicry, puromycin can affect both prokaryotic and eukaryotic ribosomes, though its effectiveness can vary between different organisms.Tissues with high metabolic activity and rapid protein synthesis may also accumulate the puromycin based radiopharmaceutical 20 .In such a case, pitfalls for image evaluation might include the presence of cancer lesions.It is therefore an excellent vector to image protein synthesis 21 but interpretation of the results will happen within the clinical context.
When comparing currently readily available PET radionuclides, generator-based gallium-68 would be a practical candidate for potential application in infection imaging.Gallium-68 support features a decentralised production also applicable to less developed hospital radiopharmacies, a frequented elution of radioactivity per day (i.e., radionuclide accessible on-demand), a generator compliance to GMP and GRPP regulations, minimal down-time, and tailored radiosynthesis properties to the tracer's requirements.It is also a very cost-efficient radionuclide 16 .Due to these advantages, this study was performed with [ 68 Ga]Ga-DOTA-puromycin and featured µPET/MRI imaging on infected mice to investigate whether this non-invasive nuclear imaging technique is beneficial to visualise mycobacterial infections in vivo.More details of the study scope are provided in Fig. 4.
Puromycin was discovered as one of the first inhibitors of protein synthesis, and the process of protein translation and the macromolecular ribosome were among the first recognized molecular targets for antibiotics.During protein synthesis both the endogenous and radiolabelled puromycin mimic the aminoacyl-tRNA and thereby bind to the free "A-site" of a ribosome.Catalytic formation of a bond between the nascent polypeptide and puromycin is followed by the release of the peptidyl-puromycin from the ribosome, as no further elongation is possible.The specific effects of puromycin have been used to investigate nascent chain length, the kinetics of chain elongation, and the identification of the effects of other antibiotics [8][9][10][11] .
We hereby present the necessary results that prove the performance of [ 68 Ga]Ga-DOTA-puromycin-PET/MRI imaging on bacterial ribosomal activity.Firstly, as missing from other literature reports, we achieved a meaningful correlation between the tracer tissue concentration (NUV) and the quantification of the mycobacterial tissue www.nature.com/scientificreports/burden (ex vivo) using histopathological analysis (ZN staining).Those results are indicative that the imaging technique accurately in line with the level of infection which allows us to suggest that radiolabelled puromycin indeed accumulates in viable bacteria by the latter described mechanism of action.Some evidence is provided to demonstrate that it could be useful for non-invasive assessment of chronic changes, however this needs further investigation.The accurate representation of the tissue pathology has been reviewed for other antibiotic-based infection imaging agents 22 .Hereby, the advantage of a well understood mechanism of action and a non-compromising radiosynthesis strategy (as applicable to [ 68 Ga]Ga-DOTA-puromycin) forms part of the motivation for potential nuclear medicine applications.However, using antibiotic derived radiopharmaceuticals might be challenged by lower imaging sensitivity hinging on the aspect that bacteria might still be exposed to antimicrobial action.Addressing the latter concern, we also provide adequate results that demonstrate lower NUV's on the [ 68 Ga]Ga-DOTA-puromycin scan compared to [ 18 F]FDG providing some evidence that it might be less sensitive than the standard [ 18 F]FDG scan.This could be detrimental to possible clinical translation for mycobacterium infection specific imaging.However, in this study, the [ 68 Ga]Ga-DOTA-puromycin NUV's calculated during acute the infection phase in mice allowed for clear visualisation of the infection site.The results also support the argument that [ 68 Ga]Ga-DOTA-puromycin can play a critical role as a research tool for the preclinical setting-given its limit of detection (lowest detectable tracer concentration over unspecific tissue concentration) is provided, measuring protein synthesis as an in vivo, non-invasive and longitudinal biomarker may provide immense value and significance, for example during drug development or monitoring disease progression.For example, puromycin reactivity using fluorescent or bioluminescent intensity was the limiting factor into study drug action against C. elegans 22 .However, the herein described [ 68 Ga]Ga-DOTA-puromycin is capable of delivering as little as 1.5 Bq gallium-68 radioactivity per fmol DOTA-puromycin to an in vivo setting, though the study setup was not directed to determine the actual in vivo detection limit for [ 68 Ga]Ga-DOTA-puromycin.
Based on the latter finding the tracer was assessed for its imaging selectivity using commonly described control animals.As the tracer accumulated equally well in S. Aureus lesions and M. Bovis lesions, results provide sufficient evidence to categorize [ 68 Ga]Ga-DOTA-puromycin as only selective for infections versus inflammation, but not more selective towards mycobacteria species over other bacteria such as the S. aureus tested in this study (pan-bacterial uptake should still be tested in future studies to further confirm selectivity).[ 68 Ga] Ga-DOTA-puromycin is therefore not a mycobacterium-specific targeting radiopharmaceutical; however, as the proven tissue response from sterile inflammation was not detectable by [ 68 Ga]Ga-DOTA-puromycin-PET/ MRI, this technique could be suggested in the research sector for example to test for drug efficacy.For general understanding a representative [ 68 Ga]Ga-DOTA-puromycin-µPET/MRI whole-body (neck-pelvis) projection 45 min post injection (Supplementary Figure S4) demonstrates expected tracer distribution in liver, spleen, and predominant renal excretion with higher radioactivity levels found in kidneys and the urinary bladder, which is characteristic for small, polar molecules.
Although a genetically different ribosome complex exists for bacteria, the actual ribosomal function with the protein synthesis and translation as a cellular mechanism is not limited to bacteria 23 .Our previous investigations confirmed [ 44 Sc]Sc-DOTA-puromycin uptake in human cancer cell lines and tumour bearing rats 15 .To date, no such explorations in humans have been reported to confirm the value of this nuclear medicine method for imaging cancer.
It is our belief that this radiopharmaceutical in alignment with sensitive PET imaging does have vast applications whenever protein synthesis is investigated as a biomarker.This may include but is not limited to infection imaging but could also be targeting malignancies and neurodegenerative disorders (via reduction) 14 .

Conclusion
Study results concerning the adequate evaluation of the [ 68 Ga]Ga-DOTA-puromycin PET/MRI imaging performance regarding infections derived from in vivo deposition of the BCG agent is presented.The accurate correlation achieved between tracer tissue concentration to gold standard methods in histopathology provides [ 68 Ga] Ga-DOTA-puromycin value and potential in preclinical research settings.The low [ 68 Ga]Ga-DOTA-puromycin imaging sensitivity during investigations of the chronic disease stage in comparison to [ 18 F]FDG-PET may be a limitation for future studies in humans which should be further investigated.

Histology
At their respective endpoint the animals were euthanized by isoflurane overdose and relevant tissues were dissected, fixed in 10% buffered formalin, and embedded in paraffin and sliced (1 μm) for histological processing.Haematoxylin-Eosin (HE; micro-anatomy), Gram (SA) and Ziehl-Neelsen staining (ZN; detecting acidfast bacilli AFB) were performed as described elsewhere [27][28][29] .Histological assessments were performed by two experienced pathologists; pathologic tissue changes and the potential bacterial load was examined for lung, myocardium, liver, spleen, and injection sites, i.e., areas of infection/ inflammation.Following chronic BCG infection lung tissue of four lung segments (left, superior, middle-posterior, and inferior) was explored.Such lung segments from 4 animals underwent randomised microscope inspection (n = 10 field of views per tissue).The level of inflammation was graded on a relative scale of 1-4 as following: grade 1-absence of inflammation; grade 2-extent of granuloma or higher number of macrophages/granulocytes; grade 3-onset of necrosis; grade 4-extended necrosis.Deriving from ZN staining, the extent of rod-shaped AFB (M.bovis) tissue manifestation was graded on a relative scale of 0-10: grade 0-absence of AFC to grade 10-maximal tissue density of AFC.An example of histology slides is provided as Supplementary Fig. 5.

Statistical analysis
Statistical analysis was performed with GraphPad Prism Version 8.0 (GraphPad Software, CA, USA).If not mentioned otherwise, the organs or tissues activity concentration was expressed as mean and standard deviation (SD).The significance of two mean values was calculated by one-or two-tailed paired and unpaired Student's-t-test with the levels of significance (p) establishing at p < 0.05 (*), < 0.01 (**) and < 0.001 (***).Spearman rank order correlation test was applied to analyse the relationship between NUV and Ziehl-Neelsen staining.

Figure 4 .
Figure 4. Study design and overview of in vivo/ex vivo experiments (Drawn on BioRender).